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Dr. Tanveer Naved

Amity Institute of Pharmacy, Amity University, Noida (U.P.) India Email: [email protected]

Address for correspondence

Access this article online www.japer.in

Standardization and Evaluation of Formulation Parameters of

Tinospora Cordifolia

Tablet

INTRODUCTION

Tinospora cordifolia also called Amrita, Giloy, Guduchi

is widely used in Ayurvedic system of medicine

“Rasayanas” to the immune system and the body

resistance against infections [1]. It is a large, glabrous, deciduous climbing shrub belonging to family

Menispermaceae is widely used in folk and Ayurvedic

system of medicine it is referred as one of the most

versatile rejuvenating herb. The species is widely

distributed in India, Malaysia, Indonesia and Thailand.

The Hindi name of the plant is Giloy, a Hindu mythological term that cites to heavenly elixir

used by Celestial beings to stay off the aging and to stay young forever [2]. The stem of T. cordifolia is

succulent with long filiform fleshy aerial roots from

the branches. The bark is creamy white to grey, deeply

left rosette like lenticels. The large numbers of

compounds have been isolated from the aerial parts and roots of T. cordifolia. Flowers are yellow, growing

in clusters from nodes. Fruits are drupes, turning red

when ripe [3]. A variety of constituents have been

isolated from different parts which includes berberin,

tinosporaside, tinosporin, tinocordifolioside,

cordifolioside A, cordifolioside B, isocolumbin, magnoflorine. It shows the presence of terpenoids,

alkaloids, lignan, carbohydrates, bitters, steroids and

glycosides. Different constituents like glycoside –

giloin and a non-glucoside – gilenin and gilosterol

have been found. The alkaloid tinosporin, tinosporic

acid and tinosporol have been identified in the leaves. Tinosporidine and sitosterol isolated from stem,

cordifol, heptacosanol and octacosonal from leaves a new furanoid diterpene – tinosporide isolated from

stems [4]. One of the most important constituent

present in stem of T. cordifolia is berberin, an

isoqunoline alkaloid having molecular formula

C20H18NO4 with molecular mass 336.36122 g/mol. It is yellow coloured alkaloid which shows strong yellow

fluorescence under U.V light. It shows various

pharmacological actions which enhances the

therapeutic efficacy of this plant. Berberin a natural

alkaloid recently used as an anticancer, antibacterial,

antipyretic, anti-inflammatory, anti malarial, antidiabetic and in treatment of cardiovascular Tinospora cordifoliaa medicinal herb used in the Indian Ayurvedic system of medicine due to their health benefits.The aim of the present work is based on the standardization, Physiochemical & Phytochemical investigation including finger print analysis by HPTLC studies. The study also includes formulation and evaluation of solid oral dosage form (tablet) from plant extract. Several improved preformulation and formulation parameters had been studied for the standardization of solid oral dosage form of tablet in order to enhance its therapeutic efficacy. Besides this antioxidant activity of the prepared formulation was evaluated by DPPH free radical scavenging method and was found to be 59µg/ml followed by estimation of total flavonol and total phenolic content i.e 29.01mg/gm and 8.12 mg/gm of a sample respectively. Antibacterial activity of the prepared formulation was also evaluated by paper disc diffusion method. Quantification of tablet were done by HPLC method and total berberin content in 500mg of tablet was found to be 218ppm.

Keywords: Tinospora cordifolia, DPPH method, Paper disc diffusion method, formulation of the solid oral dosage form.

ABST ABST ABST ABSTRACTRACTRACT RACT

Raina Mehra, Tanveer Naved*, Mahek Arora, Swati Madan

Amity Institute of Pharmacy, Amity University, Uttar Pradesh, Noida (U.P.) India

J. Adv. Pharm. Edu. & Res.

(2)

problems [5]. T. cordifolia a medicinal herb used in the Indian system of medicine due to their health benefits.

In modern medicine it is used for the treatment of

general weakness, fever, dyspepsia, dysentery,

gonorrhoea, urinary diseases, viral hepatitis and

anaemia more recently the immunomodulatory

properties, antioxidant activity, antineoplastic activity,

hypoglycemic activity, antipyretic activity, hepatoprotective activity, diuretic, anti-stress,

antihyperglycemic, antidiabetic and anti tuberculotic

activity were evaluated [6]. Hence regarding this solid

oral dosage form of tablet is prepared with improved

preformulation and formulation parameters which

prove to be useful as an antioxidant as well as antibacterial activity.

MATERIALS AND METHODS

Plant material

Dried stems of T. cordifolia were collected in the

month of January 2012 from Lahori gate. The plant

stem was authenticated by Dr. H. B. Singh, Chief Scientist & Head Raw Materials Herbarium and

Museum, NISCAIR, New Delhi vide Voucher Number

NISCAIR/RHMD/Consult/-2011-12/1917/217 Dated

January 06, 2012.

Instruments and Chemicals

Soxhlet apparatus, Round bottom flask, Heating mantle, Test tubes, China dish, Rota vapour, Silica

crucible, Ashless filter paper (Whatman no.41),

Petridish, Glass stoppered, Conical flask, Measuring

cylinder, Muffle furnace (Thermotech), Hot air oven

(NISCO), Grinding Mixer, Dessicator, Waterbath, Glass

slides, Digital electronic weighing balance (Wenser, ISO 9001: 2000 certified), TLC chamber, HPTLC plates

(Precoated aluminium sheet (10x10cm) with silica gel

60 F24 (Merk), Iodine chamber, Sprayer, HPTLC

(CAMAG), Silica gel G for thin layer chromatography

(Qualigens fine Chemicals), HCL (Merk),

Phloroglucinol (Merk) and aniline (Qualigens fine

chemicals), DPPH (Himedia Pvt Ltd), Ascorbic acid (Merk), Berberin (Sigma), FCR reagent (CDH Pvt Ltd),

Ammonium acetate (Merk specialities Pvt Ltd),

Sodium Hydrogen Orthophosphate (Fischer scientific

India Pvt Ltd), Lactose (Merk specialities Pvt Ltd), Magnesium Stereate (CDH Labs).

Physiochemical studies

Physiochemical parameters like forgein matter, total

ash, acid insoluble ash, water soluble ash, loss on

drying, extractive values like water soluble and

alcohol soluble extractive values were determined as

per Indian Pharmacopoeia

Microscopic studies

Section cutting: Dried stem were taken and soaked in

water for overnight. After that stem were cut into thin

section and stained with Phloroglucinol & HCL, I2 , KI and aniline blue on a clean glass slide and covered

with a cover slip using glycerine and observerd under microscope.

Cork cells: Loosely arranged tangentially elongated

which are broken at certain places due to the presence

of lenticels. Phelloderm the inner to the cork is an inconspicuous layer which stains yellow on treatment

with iodine and potassium iodide may be a lignified layer of phelloderm. Cortex next to phelloderm lies the cortex which consists of small compactly but

irregularly arranged polygonal parenchymatous cells.

These cortical parenchymatous cells contains

abundant starch as they stains blue with Iodine and

potassium iodide solution.

Fig 1: Transverse Section of T.cordifolia

Fig 2: Transverse Section of T.cordifolia on Treatment with Phloroglucinol & HCL

(3)

Fig 3: Transverse Section of T.cordifolia on Treatment with I2 and KI

Xylem out of which phloem is present just below the

pericyclic fibers and stained blue with differential

stain I2, KI and aniline blue. Vessels are cylindrical

and have Vascular tissue consists of phloem and simple pits with annular thickenings. The strands of

xylem are separated by 6-8 cell wide multiserate

medullary rays. Pith consists of mainly thin walled cells containing starch granules as shown in figures

given below.

Fig 4: Transverse Section of T.cordifolia on Treatment with Aniline

Preliminary Phytochemical Screening

Preliminary phytochemical screening was carried out

on the methanolic extract of stem of T.cordifolia revealed the presence of a wide range of

phytoconstituents including alkaloids, carbohydrates,

glycosides, saponins, tannins, flavonoids, steroids and

triterpenoids supporting the reason for its wide range

of biological activities [7],[8].

Fingerprint analysis of methanolic extract of T.

cordifolia stem by HPTLC

HPTLC fingerprint analysis was carried out on

methanolic extract of T. cordifolia stem with solvent

system i.e. Toluene: Ethyl acetate: Formic acid (5: 4: 1) using CAMAG HPTLC system consisting of linomat v

spotting and scanner 3. The chromatogram obtained

was studied under 254 nm, 366 nm [9].

Methodology:

100 mg of extract was weighed and dissolved in 5ml

of methanol. The sample was subjected for sonication

for 20 minutes and further be dissolved in 5ml. Samples are filtered and used for fingerprint analysis.

Precoated aluminium sheet (10x10cm, Merck,

Darmstadt, Germany) with silica gel 60 F254 of

thickness 0.2 mm were used on sample which were

applied in the form of band with the help of Linomat 3

applicator attached to HPTLC system which was programmed through winCATS, the software which

were installed with the apparatus. 1ml of each of the

sample was applied in the form of band of 3mm and

chromatogram was developed in CAMAG twin through

TLC chamber using solvent system chloroform: ethyl

acetate: formic acid (4:5:2) by using anis- aldehyde sulfuric acid as a detecting agent. The developed

chromatogram was then scanned using CAMAG TLC

Scanner 3 at 254 nm and 366 nm using slit dimension

4x0.30m.

Fig 5: Image of Methanolic extract of T. cordifolia at 254nm

(4)

Fig 6: Finger printing analysis of T. cordifolia extract at 254 nm.

Fig 8: Image of T. cordifolia after spraying

Fig 7: HPTLC Chromatographic profile (3D) of T. cordifolia extract at 254 nm

Fig 9: Image of T. cordifolia at 366nm

Fig 10: Fingerprinting analysis of T. cordifolia extract at 366 nm

(5)

Preparation of formulation

T. cordifolia tablets can be prepared by Wet granulation Method. Firstly the extract was allowed to

pass through the sieve no. 20 along with lactose

(90mg) and blend together in a mixing tray. After that

wet mass was obtained by incorporation of starch

solution (5%) to the dry mixture. The wet mass was

made into dough and forced to pass through the sieve. The wet granules obtained were allowed to dry at the

temperature of 450 C at the tray dryer for and 30

minutes. After that when granules are dried

microcrystalline Na (60mg), cross caramalose sodium

(0.9 mg), magnesium stereate (1%) and talc (1%) was

added and they are passed through the sieve and milling was done. Hence the granules were ready for

the compression to obtained tablets of about 500mg

[10].

Fig 11: HPTLC Chromatographic profile 3(D) of T. cordifolia extract at 366nm

Preformulation and formulation studies

The granules were analysed for preformulation

studies like bulk density, tapped density, [11],[12],

Hausner’s ratio, Carr’s Index and angle of repose [13]

Variuos formulation studies were studied like

hardness and friability, disintegration time,

dissolution rate of prepared formulation were

performed. According to USFDA dissolution were carried out in 3 medium i.e 0.1 N HCl, 4.6 Acetate

buffer and 6.8 phosphate buffer [14].

Quantification of prepared formulation by HPLC

Berberin content in prepared formulation were estimated by HPLC method. About 500 mg of the T.

cordifolia tablet were weighed in triplicate and

transferred it into volumetric flask and making it to

the mark with methanol. 5ml of the solution were

pipette out into clean 50 ml volumetric flasks and

made to the mark with the mobile phase acetonitril:

water (60:40), by using Column Reverse phase ODS, 250 × 4.6 mm, flow rate of about 0.5ml/min at the

wave length of about 265 nm. A calibration curve of

peak areas versus concentration of the standards was

plotted. The berberin content in the oral dosage form

was calculated using the regression equation of the

best line of fit [15].

In vitro biological activity of prepared formulation

Antioxidant activity of T. cordifolia tablet by DPPH free

radical scavenging assay. This assay is based on the

reduction of 1, 1-diphenyl-2-picrylhydrazyl (DPPH)

which is a stable free radical with purple colour. Due

to the presence of an odd electron it gives strong absorption maximum at 517 nm. Various dilutions of

the extract and prepared formulation was prepared

with methanol resulting in concentration in a range of

5µg/ml, 10 µg/ml, 20 µg/ml, 30µgm/ml, 40µg/ml,

50µg/ml, 100µg/ml, 150µg/ml. To each dilution was

added 2 ml of the prepared DPPH solution in methanol. Control was prepared simultaneously

consisting of 2ml methanol and 2ml of DPPH solution.

The prepared dilutions were then left for colour

development in the dark for 20 minutes. Finally the

absorbance of control, Standard and Sample were

measured at 517 nm against a methanol blank [16].

Total flavonol content

The total flavanol content in T. cordifolia tablet can be

determined by Aluminium chloride colorimetric

method . 10 mg of the sample was weighed and

dissolved in 1 ml of 80% methanol using a vortex

mixture (Touch Type) followed by adding to it 9 ml of

methanol and finally mixing through sonicator to prepare a sample concentration of 1mg/ml. 0.5 ml of

this solution was pipette out in a test tube to which

was added 1.5 ml of 95% methanol, 0.1 ml of 10%

(6)

aluminium chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water .The final sample solution

in the test tube was left to incubate for 30 minutes at

room temperature. The sample was prepared in

duplicate. Finally, the absorbance was measured at

415 nm against a reagent blank. The standard curve

was obtained using rutin solution in the range of 10,

20, 40, 60, 80, 100µg/ml. The results will be expressed as mg rutin/g dry weight by comparison with rutin

standard curve [17].

Total phenolic content

The total phenol content in T. cordifolia tablet was

determined by using the Standard Folin-Ciocalteu

method. 1.5ml of the sample was pipette out in a test tube to which 10.5 ml of distilled water was added

followed by addition of 0.75ml of Folin-Ciocalteu

reagent (FCR). This was left for incubation for 1-8

minutes at room temperature. Lastly to this 2.25ml of

20% Sodium carbonate solution was added and

various dilutions were obtained in concentration 100µg/ml, 200 µg/ml, 300 µg/ml, 400 µg/ml,

500µg/ml, 600µg/ml, 800µg/ml,1000µ/ml and the

dilutions were left to incubate for 2 hrs. Finally the

absorbance was measured at 765 nm against a

reagent blank. The Standard curve was obtained using

gallic acid monohydrate. The total phenol content will be expressed as gallic acid equivalent to % w/w of

sample [18].

Antibacterial activity

This method is based upon Paper disc diffusion

method which involves diffusion of an antibiotic from a filter paper disc through the solidified culture media

of a Petri dish used for study. Sample of T. cordifolia

prepared formulation were prepared in different

concentrations of about 5 µg/ml, 10 µg/ml, and 15

µg/ml with methanol. Paper disc impregnated with

sample solutions as well as antibiotic solution

(Ampicillin) were placed on to the surface of the solidified nutrient media of the plates with the help of a

sterilised wire loop. After that the plates were incubated

at 30OC at BOD incubator. After 24 hours the diameter of

zone of inhibition was measured for the evaluation of antimicrobial activity [19],[20].

RESULTS AND DISCUSSION

Physio-chemical Analysis

Physio-chemical parameters like total ash value, water

soluble ash, aid insoluble ash, loss on drying and

extractive value were shown in table 1. Table 1: Physio chemical Parametres

S. No. Parameters % w/w values

1. Forgein matter anaysis 0.12

2. Ash values

2.1 Total ash 6.12

2.2 Acid insoluble ash 1.08

2.3 Water soluble ash 1.22

3. Loss on drying 4.1

4. Extractive values

4.1 Alcohol soluble extractive value 16.03 4.2 Water solule extractive value 20.7

Preliminary Phytochemical Screening

Preliminary phytochemical screening was carried out

on the methanolic extract of stem of T.cordifolia

revealed the presence of a wide range of

phytoconstituents phytoconstituents including

alkaloids, carbohydrates, glycosides, saponins,

tannins, flavonoids, steroids and triterpenoids as shown in table no. 2.

Table 2: Phytochemical screening

S. No. Test name Methanolic extract

1. Alkaloids +

2. Carbohydrate +

3. Anthraquinone Glycosides -

4. Proteins And Amino acids -

5. Tannins And Phenolics +

6. Flvanoids +

7. Saponin -

8. Steroids +

(+) Present; (-) Absent

Fingerprint Analysis of Methanolic Extract of T.

cordifolia Stem at 254nm

HPTLC fingerprint analysis was carried out on

methanolic extract of T. cordifolia stem with solvent

(7)

using CAMAG HPTLC system. TheRf value of different compounds at 254 nm is shown in table no. 3.

Table 3: Finger print analysis of T. cordifolia at 254 nm.

S.

No. Solvent system Rf values

1.

Toulene: Ethyl acetate: Formic acid

(5:4:1)

0.38,0.44,0.50,0.53, 0.58,0.65,0.67 (7 Spots)

Fingerprint Analysis of Methanolic Extract of T.

cordifolia Stem at 366nm as shown in table no. 4.

Table 4: Finger print analysis of T. cordifolia at 366 nm.

S.

No. Solvent system Rf values

1.

Toulene: Ethyl acetate: Formic acid

(5:1:15)

0.33, 0.38, 0.44, 0.49, 0.56, 0.60, 0.65, 0.75, 0.82, 0.86

(10 Spots)

Preformulation Studies for the Preparation of Tablet

Preformulation parameters like bulk density, tap

density, Carr’s index, Hausner’s ratio and angle of

repose were carried out. The granules showed excellent flow property as shown in table no. 5.

Table 5: Preformulation parameters

S. No. Parameters Result

1. Bulk density 0.421 (gm/ml) 2. Tapped density 0.512 (gm/ml) 3. Hausner’s ratio 1.21 4. Carr’s index 17.7 5. Angle of repose 32.18 (θ)

Formulation Studies of Prepared Formulation Formulation studies like hardness and friability,

disintegration test and dissolution test were studied

for the evaluation of tablet and shown in table no. 6

Table 6: Formulation parameters

S. No. Parameters Results

1. Hardness 3.9 (kg/cm2)

2. Friability 0.52 %

3. Disintegration 13 minutes 20 Secs 4. Dissolution

a. 0.1 NHCl 76.54 %

b. 4.6 Phosphate buffer 73.1% c. 6.8 Acetate buffer 75.8%

Quantification of 500mg of tablet by HPLC

A sharp peak of berberin was obtained by using

mobile phase as acetonitrile: water (40:60). At a

retention time of 5.6 at 265 nm. Berberin content in

500 mg of tablet was found to be 218 ppm as shown in

table no. 7.

Table 7: Estimation of berberin content in 500 mg of prepared tablet

S. No. Formulation Berberin Content in 500mg of Tablet

1. Tablet 218ppm

Antioxidant activity of prepared formulation Antioxidant activity of prepared formulation was

assessed by DPPH free radical scavenging assay. The formulation showed potent antioxidant activity with

an inhibitory concentration (IC50) of 59µg /ml .

Ascorbic acid used as a standard showed an IC50 value

of 6.12µg/ml as shown in table no.8

Fig 12: Standard curve of Ascorbic acid

Fig 13: Free radical assay curve for tablet

(8)

Table 8: IC 50 value of prepared formulation

S. No. Sample IC50 value

1. Std Ascorbic acid 6.12 µg/ml

2. Tablet 59 µg/ml

Estimation of total flavonol content

The total flavonol content in prepared formulation

was found to be 29.01 mg/gm as shown in table no.9.

Rutin was used as a standard.

Fig 14: Standard curve for Rutin

Table 9: Result of Total Flavonol Content in T. cordifolia Tablet

S.

No. Formulation

Total Flavonol Content (mgRE/g of sample)

1. Tablet 29.01

Estimation of total phenolic content

The total phenolic content in prepared formulation

was found to be 8.12mg/gm as shown in table no. 10.

Standard curve for Gallic acid was obtained.

Fig 15. Standard curve for Gallic acid.

Table 10: Result of Total Phenol Content in T. cordifolia Tablet

S.

No. Formulation

Total Phenol Content (mgGAE/gof sample)

1. Tablet 8.12

Antibacterial activity of Prepared formulation The prepared formulation of T. cordifolia extract was

screened for their antibacterial activity by paper disc diffusion method. Though the highest activity was

observed against E.coli having Zone of Inhibition of 2.1

cm at the concentration of about 15µg/ml. Moreover,

activity was quite reasonable and concentration-

dependent in B. subtilis bacteria having Zone of

Inhibition of diameter 1.2 cm at the concentration of about 15µg/ml . However, the standard drug

ampicillin at the concentration of 5 µg/ml showed

maximum activity against B. subtilis at the

concentration of about 15 µg/ml having Zone of

Inhibition of diameter of about 3.3cm as shown in

table no.11.

Fig 16: Zone of Inhibition of E.coli

(9)

Table 11: Results of Zone of Inhibition in T. cordifolia Tablet with Tetracycline by Paper disc diffusion method.

S. No Formulation Concentration(µg/ml) E.coli (Zone of inhibition) (cm) B.subtilis (Zone of Inhibition) (cm)

1. Tablet 5 µg/ml 0 0

10 µg/ml 0 0

15 µg/ml 2.1 1.2

2. Tetracycline 5µg/ml 3.2 3.3

CONCLUSION

The present study provides valuable information

regarding the identification and authentication of the

plant T. cordifolia along with the development of the

solid oral dosage form with improved formulation

parameters. Antioxidant rich plants serve as source of nutraceuticals that alleviate the oxidative stress and

therefore prevent or reduce the onset of degenerative

diseases. Therefore antioxidant activity of prepared

formulation was evaluated by DPPH free radical

scavenging assay. Antibacterial activity of the

formulation was performed against E.coli and B.subtilus which clearly claimed its effects against

several infections, inflammations and several other

therapeutic benefits for human health. The present

study justify the use of prepared formulation of T.

cordifolia tablet in treatment of various infectious

diseases and as a source of nutraceuticals in order to reduce oxidative stress with consequent health

benefits. So further work could be done for the

isolation and purification of important compounds

from this plant which will allow the scientific

community to utilise as an accessible alternative for

the production of synthetic antibiotics. It shows many pharmacological activities as well. Hence this provides

us a great scope of investigation regarding future

prospects also.

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How to cite this article: Raina Mehra, Tanveer Naved*,

Mahek Arora, Swati Madan; Standardization and Evaluation of Formulation Parameters of Tinospora Cordifolia Tablet: An Overview; J. Adv. Pharm. Edu. & Res. 2013: 3(4): 440-449.

Figure

Fig 1: Transverse Section of T.cordifolia
Fig 5: Image of Methanolic extract of T. cordifolia at 254nm
Fig 7: HPTLC Chromatographic profile (3D) of T.
Fig 11: HPTLC Chromatographic profile 3(D) of T. cordifolia extract at 366nm
+4

References

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Highest values of D A 665u were recorded for the humic acids isolated from the soil samples additionally polluted with PAHs after 180 days of incubation.. Humic acids of

Although both sets of guidelines focus on the importance of consistency, support for expert users, feedback, robustness, and support recognition rather than recall, the

The qualifying aspects of the TNGB can be summarised as follows: (i) the rigorous selection criteria adopted by Telethon for choosing the Biobanks to receive funding along with